The long term objective of the project remains the same: to understand the plasticity of the human genome at the molecular level with special emphasis on the evolution of multigene families. Our molecular analysis of the haptoglobin gene cluster in humans and in primates has been highly successful to date in demonstrating the complex history of the human genome, even when only a limited region is examined. The main thrust of the next phase of our study is to test by direct experiments the hypotheses we have developed during these past studies of the evolution of the haptoglobin gene cluster. Aim 1 is to evaluate the recombinational properties of specific DNA sequences in vitro. We will determine whether recombinational "hot spots", such as we have hypothesized in the haptoglobin gene cluster and other investigators have described elsewhere, affect the frequency of recombination in cell culture. To do this, we will score recombination in mouse embryonic stem (ES) cells between exogenous DNA and DNA previously introduced into the hypoxanthine phospho-ribosyl transferase (Hprt) locus. Aim 2 is to evaluate the effects of insertion of various elements on the expression of nearby genes. We will test whether or not the retrovirus- like sequence in the first intron of the human haptoglobin related gene (HPR) is, as we have hypothesized, responsible for inactivation of this gene. ES cells will be used to determine the effects on gene-expression of elements inserted into an intron of the Hprt gene. Aim 3 is to investigate the overall function of the haptoglobin and haptoglobin related genes using the mouse. Despite the fact that haptoglobin was discovered over 50 years ago, its overall function is still not known. We will use gene targeting to generate mice having no haptoglobin gene, and mice in which the human HP2 or HPR gene has replaced the mouse gene in its normal chromosomal context. We will also generate mice carrying a copy of the HPR gene with DNA fragments, identified in specific aim 2 as having significant effects on haptoglobin expression, inserted into intron 1. In this way, we will test the hypothesis concerning haptoglobin gene expression that we have developed in the course of our evolutionary studies.